Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional programs.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Vesiclepedia 2024: an extracellular vesicles and extracellular particles repository.

Vesiclepedia (http://www.microvesicles.org) is a free web-based compendium of DNA, RNA, proteins, lipids and metabolites that are detected or associated with extracellular vesicles (EVs) and extracellular particles (EPs). EVs are membranous vesicles that are secreted ubiquitously by cells from all domains of life from archaea to eukaryotes. In addition to EVs, it was reported recently that EPs like exomeres and supermeres are secreted by some mammalian cells. Both EVs and EPs contain proteins, nucleic acids, lipids and metabolites and has been proposed to be implicated in several key biological functions. Vesiclepedia catalogues proteins, DNA, RNA, lipids and metabolites from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 3533 EV studies, 50 550 RNA entries, 566 911 protein entries, 3839 lipid entries, 192 metabolite and 167 DNA entries. Quantitative data for 62 822 entries from 47 EV studies is available in Vesiclepedia. The datasets available in Vesiclepedia can be downloaded as tab-delimited files or accessible through the FunRich-based Vesiclepedia plugin.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

Bovine milk-derived extracellular vesicles enhance doxorubicin sensitivity in triple negative breast cancer cells by targeting metabolism and STAT signalling.

Metastatic triple-negative breast cancer (TNBC) has a low 5-year survival rate of below 30% with systemic chemotherapy being the most widely used treatment. Bovine milk-derived extracellular vesicles (MEVs) have been previously demonstrated to have anti-cancer attributes. In this study, we isolated bovine MEVs from commercial milk and characterised them according to MISEV guidelines. Bovine MEVs sensitised TNBC cells to doxorubicin, resulting in reduced metabolic potential and cell-viability. Label-free quantitative proteomics of cells treated with MEVs and/or doxorubicin suggested that combinatorial treatment depleted various pro-tumorigenic interferon-inducible gene products and proteins with metabolic function, previously identified as therapeutic targets in TNBC. Combinatorial treatment also led to reduced abundance of various STAT proteins and their downstream oncogenic targets with roles in cell-cycle and apoptosis. Taken together, this study highlights the ability of bovine MEVs to sensitise TNBC cells to standard-of-care therapeutic drug doxorubicin, paving the way for novel treatment regimens.

Proteomics analysis of C2C12 myotubes treated with atrophy inducing cancer cell-derived factors.

Cancer-associated cachexia is a wasting syndrome that results in dramatic loss of whole-body weight, predominantly due to loss of skeletal muscle mass. It has been established that cachexia inducing cancer cells secrete proteins and extracellular vesicles (EVs) that can induce muscle atrophy. Though several studies examined these cancer-cell derived factors, targeting some of these components have shown little or no clinical benefit. To develop new therapies, understanding of the dysregulated proteins and signaling pathways that regulate catabolic gene expression during muscle wasting is essential. Here, we sought to examine the effect of conditioned media (CM) that contain secreted factors and EVs from cachexia inducing C26 colon cancer cells on C2C12 myotubes using mass spectrometry-based label-free quantitative proteomics. We identified significant changes in the protein profile of C2C12 cells upon exposure to C26-derived CM. Functional enrichment analysis revealed enrichment of proteins associated with inflammation, mitochondrial dysfunction, muscle catabolism, ROS production, and ER stress in CM treated myotubes. Furthermore, strong downregulation in muscle structural integrity and development and/or regenerative pathways were observed. Together, these enriched proteins in atrophied muscle could be utilized as potential muscle wasting markers and the dysregulated biological processes could be employed for therapeutic benefit in cancer-induced muscle wasting.

Proteomic analysis of the small extracellular vesicles and soluble secretory proteins from cachexia inducing and non-inducing cancer cells.

Cancer cachexia is a wasting syndrome characterised by the loss of fat and/or muscle mass in advanced cancer patients. It has been well-established that cancer cells themselves can induce cachexia via the release of several pro-cachectic and pro-inflammatory factors. However, it is unclear how this process is regulated and the key cachexins that are involved. In this study, we validated C26 and EL4 as cachexic and non-cachexic cell models, respectively. Treatment of adipocytes and myotubes with C26 conditioned medium induced lipolysis and atrophy, respectively. We profiled soluble secreted proteins (secretome) as well as small extracellular vesicles (sEVs) released from cachexia-inducing (C26) and non-inducing (EL4) cancer cells by label-free quantitative proteomics. A total of 1268 and 1022 proteins were identified in the secretome of C26 and EL4, respectively. Furthermore, proteomic analysis of sEVs derived from C26 and EL4 cancer cells revealed a distinct difference in the protein cargo. Functional enrichment analysis using FunRich highlighted the enrichment of proteins that are implicated in biological processes such as muscle atrophy, lipolysis, and inflammation in both the secretome and sEVs derived from C26 cancer cells. Overall, our characterisation of the proteomic profiles of the secretory factors and sEVs from cachexia-inducing and non-inducing cancer cells provides insights into tumour factors that promote weight loss by mediating protein and lipid loss in various organs and tissues. Further investigation of these proteins may assist in highlighting potential therapeutic targets and biomarkers of cancer cachexia.

Pep2Graph: A standalone tool to analyse proteolytic cleavages by proteases from gel-based mass spectrometry data.

Proteases are enzymes that regulate substrates via proteolytic activation and coordinate essential cellular functions including DNA replication, DNA transcription, cell proliferation, differentiation, migration and apoptosis. However, techniques to identify proteolytic events in a high-throughput manner is limited. PROtein TOpography and Migration Analysis Platform (PROTOMAP) is a technique that relies on mass spectrometry-based proteomics to globally identify the shifts in the in-gel migration of proteins and their corresponding fragments that are obtained by proteolysis. However, user-friendly software tool to analyse the proteomic data to identify proteolytic events is needed. Here, we report Pep2Graph, a user-friendly standalone tool that integrates peptide sequence information from in-gel proteomics and presents the data as two-dimensional peptographs with in-gel migration, sequence coverage and MS/MS spectra counts. Pep2Graph (http://www.mathivananlab.org/Pep2Graph) allows users to utilize in-gel proteomics data to study proteolytic events that may play a significant role in normal physiology and pathology.

Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells.

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.

The Role of Post-Translational Modifications in Targeting Protein Cargo to Extracellular Vesicles.

Extracellular vesicles (EVs) are naturally occurring nanoparticles that contain proteins and nucleic acids. It is speculated that cells release EVs loaded with a selective cargo of proteins through highly regulated processes. Several proteomic and biochemical studies have highlighted phosphorylated, glycosylated, ubiquitinated, SUMOylated, oxidated and palmitoylated proteins within the EVs. Emerging evidences suggest that post-translational modifications (PTMs) can regulate the sorting of specific proteins into EVs and such proteins with specific PTMs have also been identified in clinical samples. Hence, it has been proposed that EV proteins with PTMs could be used as potential biomarkers of disease conditions. Among the other cellular mechanisms, the endosomal sorting complex required for transport (ESCRT) is also implicated in cargo sorting into EVs. In this chapter, various PTMs that are shown to regulate protein cargo sorting into EVs will be discussed.

Engineering Extracellular Vesicles for Cancer Therapy.

Extracellular vesicles (EVs) are lipid bilayer containing nanovesicles that have a predominant role in intercellular communication and cargo delivery. EVs have recently been used as a means for drug delivery and have been depicted to elicit no or minimal immune response in vivo. The stability, biocompatibility and manipulatable tumour homing capabilities of these biological vessels make them an attractive target for the packaging and delivery of drugs and molecules to treat various diseases including cancer. The following chapter will summarise current EV engineering techniques for the purpose of delivering putative drugs and therapeutic molecules for the treatment of cancer. The relevance of EV source will be discussed, as well as the specific modifications required to manufacture them into suitable vehicles for molecular drug delivery. Furthermore, methods of EV cargo encapsulation will be evaluated with emphasis on intercellular coordination to allow for the effective emptying of therapeutic contents into target cells. While EVs possess properties making them naturally suitable nanocarriers for drugs and molecules, many challenges with clinical translation of EV-based platforms remain. These issues need to be addressed in order to harness the true potential of the EV-based therapeutic avenue.

Introduction to the Community of Extracellular Vesicles.

Since the discovery that extracellular vesicles (EVs) mediate intercellular communication, there is an exponential increase in the interest on EVs, especially in pathological settings. EVs are membranous vesicles that are secreted by various cell types and the release of EVs is conserved in every prokaryotic and eukaryotic organism tested to date. These vesicles were initially thought to be garbage disposal vehicles and subsequent studies over the past 4 decades have attributed several functional roles to EVs, some of which are critical for homeostasis. The molecular cargo of nucleic acids, proteins, lipids and metabolites packaged in EVs often mirror the host cells phenotypic status. EVs can be taken up by recipient cells and upon uptake, EVs through its molecular cargo, can induce a cascade of signal transduction events in recipient cells. EVs are categorised into several subtypes depending on their biogenesis and secretion. Due to several subtypes, differing sizes within a subtype and varying cargo, EVs are heterogenous in nature and the biophysical and biochemical properties of EVs often overlap between EV subtypes. Hence, it is important to be cautious when selecting the method of EV isolation and characterisation. This chapter provides a brief introduction to EVs and their subtypes.

Biogenesis of Extracellular Vesicles.

Extracellular vesicles (EVs) refer to vesicles that are released by cells into the extracellular space. EVs mediate cell-to-cell communication via delivery of functional biomolecules between host and recipient cells. EVs can be categorised based on their mode of biogenesis and secretion and include apoptotic bodies, ectosomes or shedding microvesicles and exosomes among others. EVs have gained immense interest in recent years owing to their implications in pathophysiological conditions. Indeed, EVs have been proven useful in clinical applications as potential drug delivery vehicles and as source of diagnostic biomarkers. Despite the growing body of evidence supporting the clinical benefits, the processes involved in the biogenesis of EVs are poorly understood. Hence, it is critical to gain a deeper understanding of the underlying molecular machineries that ultimately govern the biogenesis and secretion of EVs. This chapter discusses the current knowledge on molecular mechanisms involved in the biogenesis of various subtypes of EVs.

Exomeres: A New Member of Extracellular Vesicles Family.

Extracellular vesicles (EVs) are described as membranous vesicles that are secreted by various cell types. EVs can be categorised as exosomes, ectosomes, apoptotic bodies, large oncosomes and migrasomes. EVs are heterogeneous in nature according to their origin, mode of release, size, and biochemical contents. Herein, we discuss a recently discovered subpopulation of EVs called 'exomeres'. Unlike the other subtypes of EVs, exomeres are defined as non-membranous nanovesicles with a size ≤50 nm. They can be isolated using asymmetric-flow field-flow fractionation as well as ultracentrifugation. The cargo of exomeres are beginning to be unravelled and are highlighted to be enriched with proteins implicated in regulating metabolic pathways. Consistent with other types of EVs, exomeres also contain nucleic acids and lipids which can be delivered to recipient cells. These discoveries highlight the complex heterogeneity of EVs and thereby necessitates further attention to understand the nature of each subpopulation more exclusively. Overall, this chapter describes the current knowledge on exomeres.

Extracellular Vesicles Regulate Cancer Metastasis.

Metastatic cancer is a complex disease associated with poor prognosis and accounts for the majority of cancer related deaths. To date, many of the molecular mechanisms driving metastatic disease remain elusive and require further investigation for the development of effective treatment strategies. Recent studies have shown that extracellular vesicles (EVs) can be exploited by tumors to assist in cancer cell growth, proliferation, migration, invasion and metastasis. Cancer cells have proven efficient in educating fibroblasts, within their microenvironment, to secrete EVs as communicative vessels for mediating phenotypic changes in recipient cells. Using this vesicular delivery system, cancer cells can establish a new metastatic niche within distant sites, away from the primary tumor, thus favoring cancer progression. These findings demonstrate the availability of a new route for therapeutic intervention in the inhibition of cancer dissemination. Although, several approaches to target cancer cell secretion of EVs are detailed in the literature, there is still no defined way to currently apply them in clinical settings. Hence, further studies are required to unravel the molecular mechanisms of metastasis - governed by the establishment and release of cancer associated EVs.

Extracellular Vesicles in Metabolism and Metabolic Diseases.

As living organisms constantly need energy to maintain and perform cellular functions, metabolism plays a vital role in producing the required energy to execute these processes. Hence, various metabolic pathways are highly regulated and disruption in critical pathways can result in the onset of multiple disorders such as hypertension, diabetes, obesity, and dyslipidaemia. Extracellular vesicles (EVs) are membrane-bound nanosized vesicles that are known to be secreted by various cell types into their respective extracellular environment. EVs have been implicated in cell-to-cell communication via mediating cellular signaling and can functionally impact recipient cells with the transport of bioactive proteins, nucleic acids, lipids and cellular metabolites. Recently, several studies have highlighted the role of EVs in metabolism. Alterations in the plasma derived EV concentration and their cargo in patients with metabolic disorders have been reported by multiple studies, further proposing EVs as a potential source of disease biomarkers. The following chapter will discuss the functional significance of EVs in metabolic diseases and the processes by which EVs act as cellular messengers to reprogram the metabolic machinery in recipient cells.

Are Dietary Extracellular Vesicles Bioavailable and Functional in Consuming Organisms?

It has been well established that diet influences the health status of the consuming organism. Recently, extracellular vesicles (EVs) present in dietary sources are proposed to be involved in cross-species and kingdom communication. As EVs contain a lipid bilayer and carry bioactive cargo of proteins and nucleic acids, they are proposed to survive harsh degrading conditions of the gut and enter systemic circulation. Following the bioavailability, several studies have supported the functional role of dietary EVs in various tissues of the consuming organism. Simultaneously, multiple studies have refuted the possibility that dietary EVs mediate cross-species communication and hence the topic is controversial. The feasibility of the concept remains under scrutiny primarily owing to the lack of significant in vivo evidence to complement the in vitro speculations. Concerns surrounding EV stability in the harsh degrading gut environment, lack of mechanism explaining intestinal uptake and bioavailability in systemic circulation have impeded the acceptance of their functional role. This chapter discusses the current evidences that support dietary EV-based cross species communication and enlists several issues that need to be addressed in this field.

BCL-2 family protein BOK is a positive regulator of uridine metabolism in mammals.

BCL-2 family proteins regulate the mitochondrial apoptotic pathway. BOK, a multidomain BCL-2 family protein, is generally believed to be an adaptor protein similar to BAK and BAX, regulating the mitochondrial permeability transition during apoptosis. Here we report that BOK is a positive regulator of a key enzyme involved in uridine biosynthesis; namely, uridine monophosphate synthetase (UMPS). Our data suggest that BOK expression enhances UMPS activity, cell proliferation, and chemosensitivity. Genetic deletion of Bok results in chemoresistance to 5-fluorouracil (5-FU) in different cell lines and in mice. Conversely, cancer cells and primary tissues that acquire resistance to 5-FU down-regulate BOK expression. Furthermore, we also provide evidence for a role for BOK in nucleotide metabolism and cell cycle regulation. Our results have implications in developing BOK as a biomarker for 5-FU resistance and have the potential for the development of BOK-mimetics for sensitizing 5-FU-resistant cancers.

Pannexin-1 channel regulates nuclear content packaging into apoptotic bodies and their size.

Apoptotic bodies (ApoBDs), which are large extracellular vesicles exclusively released by apoptotic cells, possess therapeutically exploitable properties including biomolecule loadability and transferability. However, current limited understanding of ApoBD biology has hindered its exploration for clinical use. Particularly, as ApoBD-accompanying cargoes (e.g., nucleic acids and proteins) have major influence on their functionality, further insights into the mechanism of biomolecule sorting into ApoBDs are critical to unleash their therapeutic potential. Previous studies suggested pannexin 1 (PANX1) channel, a negative regulator of ApoBD biogenesis, can modify synaptic vesicle contents. We also reported that trovafloxacin (a PANX1 inhibitor) increases proportion of ApoBDs containing DNA. Therefore, we sought to define the role of PANX1 in regulating the sorting of nuclear content into ApoBDs. Here, using flow cytometry and label-free quantitative proteomic analyses, we showed that targeting PANX1 activity during apoptosis, via either pharmacological inhibition or genetic disruption, resulted in enrichment of both DNA and nuclear proteins in ApoBDs that were unexpectedly smaller in size. Our data suggest that PANX1, besides being a key regulator of ApoBD formation, also functions as a negative regulator of nuclear content packaging and modulator of ApoBD size. Together, our findings provide further insights into ApoBD biology and form a novel conceptual framework for ApoBD-based therapies through pharmacologically manipulating ApoBD contents.

Vesiclepedia 2019: a compendium of RNA, proteins, lipids and metabolites in extracellular vesicles.

Extracellular vesicles (EVs) are membranous vesicles that are released by both prokaryotic and eukaryotic cells into the extracellular microenvironment. EVs can be categorised as exosomes, ectosomes or shedding microvesicles and apoptotic bodies based on the mode of biogenesis. EVs contain biologically active cargo of nucleic acids, proteins, lipids and metabolites that can be altered based on the precise state of the cell. Vesiclepedia (http://www.microvesicles.org) is a web-based compendium of RNA, proteins, lipids and metabolites that are identified in EVs from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 1254 EV studies, 38 146 RNA entries, 349 988 protein entries and 639 lipid/metabolite entries. Vesiclepedia is publicly available and allows users to query and download EV cargo based on different search criteria. The mode of EV isolation and characterization, the biophysical and molecular properties and EV-METRIC are listed in the database aiding biomedical scientists in assessing the quality of the EV preparation and the corresponding data obtained. In addition, FunRich-based Vesiclepedia plugin is incorporated aiding users in data analysis.

Arrestin-Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid-bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin-domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1-deficient (Arrdc1-/- ) mice. Nanoparticle tracking analysis-based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.